Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its implications on cell behavior. This project has been a part of the Program Project Grant since its inception in 1990. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl terminal (CT) domain and the second half of the cytoplasmic loop of Cx43. We propose that specific peptides can be designed to interfere with this interaction and modulate Cx43 regulation. We have used a highthroughput phage display assay to identify peptides that bind to Cx43CT. We found that Cx43CT preferentially bound to peptides containing a specific "RXP" motif. Several RXP peptides bind to Cx43CT with micromolar affinity, cause structural modifications in the Cx43CT domain and modify Cx43 channel function and regulation. It is the goal of this proposal to further characterize these peptides and search for analogous sequences which may modulate the function of Cx43 channels. The specific aims are: Aim 1.) To characterize the high-order structure of RXP/Cx43CT complexes. Aim 2.) To characterize the effects of RXP- 1 and RXP-4 on the regulation of Cx43 channels. Aim 3.) To identify the structural constraints mediating the binding of RXP peptides to Cx43CT and their functional effects on Cx43 channels. We will use an iterative combination of spectroscopic methods (Nuclear Magnetic Resonance, Surface Plasmon sonance), proteomics approaches (high-throughput biased phage display;peptide synthesis) and functional assays to develop a structural-activity correlate toward peptides with higher affinity and selective functional effects. These peptides will potentially be probes for the manipulation of Cx43 channels and their regulation. These studies will lead to a better understanding of the role of connexins in health and disease.